This proposal is based on a well-characterized system that shows the existence of intermediate stages in the progression of a normal cell to cancer. The intermediate stage cells (I-cell) are transformed yet sensitive to normal host protective mechanisms. These cells, unlike cancer cells, do not form tumors in normal mice, but do form tumors in immunosuppressed mice. The protective mechanism consists of two components: (i) natural cytotoxic (NC) cell activity and (ii) an immune response to neo-antigen(s) on the I-cell. The sensitivity of the I-cell to NC cell lysis has been studied in detail and shown to be due to a loss of the ability to repair damage inflicted by the NC effectors. Normal cells and cancer cells are able to repair the damage efficiently and survive. The study of these processes, and other properties of cells progressing from normal to I-cells to cancer cells has benefitted tremendously from the isolation of stable cell lines with the phenotypic characteristic of each stage. These lines provide the basis for the molecular analysis of the transition that is proposed in this application. The overall goal is to define the molecular events required for a cell to progress through intermediate stages to cancer. To examine these stages the genes that control the advancement of cells from the normal to malignant phenotype will be isolated and characterized. Differences in gene expression between the stepwise variants will be determined using subtractive hybridization to select cDNA clones representing differentially expressed mRNAs. The characteristics of the cDNAs (pattern of expression, sequence and relation to known genes) will be determined. The proposed studies should provide new information on the genes and molecules involved in the susceptibility of intermediate stage cells to host protective mechanisms as well as those genes involved in the transformed phenotype. These experiments will also provide the basis for diagnostic test development using DNA probes or specific antibodies to identify preneoplastic stages. Eventually, it is hoped that further research based on these studies will be able to discover ways to eliminate the cancer initiation process.